This invention relates to methods for diagnosis of cellular senescence and immortalization.
The following is a general description of art relevant to the present invention. None is admitted to be prior art to the invention.
Michael D. West et al., entitled "Therapy and Diagnosis of Conditions Related to Telomere Length and/or Telomerase Activity," U.S. application Ser. No. 08/151,477, filed Nov. 12, 1993, and PCT WO 93/04546 filed Nov. 25, 1993, both hereby incorporated by reference, generally describe art which relates to cellular senescence, and theories or hypotheses which explain such aging and the mechanisms by which cells escape senescence and immortalize. West et al. discusses the use of telomerase activity detection for diagnosis of various cancerous conditions.
West et al., U.S. application Ser. No. 08/151,477, filed Nov. 12, 1993, describes methods by which telomerase activity can be used to determine whether a cell is immortalized. One such method (example 21) involves providing appropriate primers and using the polymerase chain reaction to determine the presence of telomerase activity in the cells.
In this method, West et al. rely upon the ability of telomerase to template and catalyze the synthesis of telomeric sequence onto a single-stranded oligodeoxynucleotide (oligo) substrate. The conventional assay for this activity uses an oligo of known sequence as substrate, radioactive deoxynucleotide triphosphate (dNTP) for labeling, and sequencing gel for resolution and display of the products. Since telomerase stalls and can release the DNA after adding the first G in the T.sub.2 AG.sub.3 repeat, the characteristic pattern of products is a six nucleotide ladder of extended oligo substrate. The phase of the repeats depends on the 3' end sequence of the oligo substrate; telomerase recognizes where it is in the repeat and synthesizes accordingly to yield contiguous repeat sequence. Although telomeric sequence oligos are the most efficient in vitro substrates, telomerase will also synthesize T.sub.2 AG.sub.3 repeats onto non-telomeric oligos.
The basis of the assay of West et al. is application of the polymerase chain reaction (PCR) for specific amplification of the in vitro products of telomerase. The telomerase reaction portion of the assay is unchanged. The telomerase products (the nested set of one to hundreds of six nucleotide telomeric repeats added to the oligo substrate) serve as the templates for PCR amplification. Exponential amplification is achieved by a PCR primer set consisting of a downstream primer complementary to telomeric repeats and an upstream primer composed of sequence from the oligo substrate. In fact, the oligo substrate (typically an 18-mer) itself serves as the upstream primer and since a standard telomerase reaction contains 1 .mu.M oligo substrate of which less than 1% is extended by telomerase, there is no need to add more for the PCR reaction. A downstream primer of comparable length is used. A stringent annealing temperature in the PCR cycle ensures specific primer binding which results in preservation of the six nucleotide ladder in the PCR products, reflective of the template population.
West et al. determined a downstream primer which they designated CX. CX is composed of sequence complementary to three imperfect telomeric repeats and one perfect repeat (5' (CCCTTA).sub.3 CCCTAA-3') (SEQ ID NO:1). The single nucleotide difference in three of the repeats compromises the capacity of CX to anneal to the 3' end of M2 (which contains 5 of 6 nucleotides of a telomeric repeat). Under optimized conditions, West et al. found that M2 and CX alone in a PCR reaction set up at room temperature and then subjected to 27 cycles of 95.degree., 50.degree., and 72.degree. produced no PCR products.
As yet another measure to prevent primer interaction and non-specific amplification, West et al. also suggested that the hot start method be utilized. In this technique, CX was dried at the bottom of the tube and then covered with a wax barrier. All other PCR reaction components were combined in the tube above the wax barrier, and the tube was placed in the thermal cycler. With this set-up, CX did not appear in the PCR reaction until the wax melted (about 60.degree. C.) during the first cycle, preventing CX interaction with any other reaction component at a temperature below the annealing temperature.
West et al. suggested that since both telomerase and Taq polymerase are DNA synthesizing enzymes with similar reaction components, the assay could be further streamlined by combining the activities in a single reaction. A single tube protocol was achieved. In this protocol, CX oligo is isolated by wax barrier for hot start of the PCR. All other reaction components are combined above the wax barrier including the telomerase oligo substrate/upstream primer M2, the telomerase extract, and Taq polymerase. PCR buffer and deoxynucleotide conditions allow sufficient telomerase product generation in 10 minutes at room temperature. The tubes are then simply placed in the thermal cycler for PCR. West et al. showed that specific amplification of telomerase products under these conditions occurs if and only if the oligo substrate M2 has been extended with three or more T.sub.2 AG.sub.3 repeats.
West et al. also determined that a correlation exists between telomerase activity and the tumor cell phenotype.